The advent of methods for the production of functional antibody fragments in recombinant bacteria (1, 2) has opened the way to genetic manipulation of antibody genes. As a result of this development, genetic engineering is increasingly being used to generate antibodies for particular applications. Examples of this methodology are antibody chimerization (3) and humanization (4). These processes produce antibodies better suited to human therapeutic applications than their typically murine progenitors, by altering parts of the antibody molecule which are recognized as foreign by the human immune system.
Although genetic engineering can be used to make almost any desired change in an antibody structure, it cannot as yet provide solutions to some of the most pressing problems in antibody generation. For example, a common goal is affinity maturation, the optimization of antibody-antigen interaction. In most cases, the three dimensional structure of the antigen is unknown, which makes rational design of the antibody binding site so as to increase affinity almost impossible. Therefore, the ability to alter antibody genes at will does not necessarily help in affinity maturation. Similarly, genetic engineering does not allow generation of human antibodies, which are desirable for therapeutic purposes, but which cannot, for ethical reasons, be generated by immunizing volunteers. Problems such as these demand a new approach to antibody generation.
A promising approach relies on searching for antibodies having the desired properties within a large collection (library) of variants. Methods for generating such antibody libraries, and searching through them, have recently been developed (see, for example, (5) and (6) and references therein). This approach is attractive because:
It relies on strong binding of the antibody to a target antigen, and is therefore well-suited to affinity maturation. PA1 Antibodies which are not available by immunization (for example, human antibodies) can be accessed.
Currently, the most efficient approach to antibody affinity maturation uses the following technique. Antibody genes from a suitable source (such as, for example, human peripheral blood lymphocytes, or human bone marrow) are cloned in bacteriophage in such a way that antibody fragments are displayed as fusion proteins on the surface of the phage (7). The phages are produced from bacterial host cells, generating a "phage library" in which every phage contains the genetic information for the antibody variant displayed on its surface. This library can be searched for antibodies which bind to the target antigen.
Locating the highest affinity antibodies within such a library is typically performed by screening: a physical process in which high affinity antibodies are separated from others through their ability to bind to immobilized antigen. The screening process involves immobilizing the target substance on a solid support and performing affinity chromatography or "panning" (8) of the phage library. Those antibodies with high affinity for the immobilized antigen are thereby enriched, and their numbers can be increased by propagation of the specifically eluted phage in bacterial host cells.
Initial results show that antibodies with moderate target affinity can be generated in this way. Higher affinities can, in principle, be obtained by performing random mutagenesis on the antibodies isolated in the first screening, and repeating the process one or more times. For example, random mutagenesis of antibody-encoding genes (9), and random shuffling of the genes encoding the component chains of the antibody (10) have been used to generate new antibodies with increased affinity or altered specificity, which have been located within the mutant libraries by screening.
Although a useful tool for antibody affinity maturation, library screening suffers from two main disadvantages. First, the probability of finding a high affinity antibody is related to the size of the library. Due to technical limitations associated with the efficiency with which bacterial cells can be transformed by plasmid DNA, libraries rarely contain more than 10.sup.8 members. This is not large enough to contain high affinity antibodies routinely. Second, phage isolated by screening must be used to re-infect bacteria if the system is to be run over multiple rounds. The overall process is therefore discontinuous, and as the physical separation and re-infection steps are time-consuming and labour intensive, the method is not well suited to multiple rounds of screening.
These disadvantages are overcome by the present invention, which provides for an artificial method of antibody optimization based on biological selection rather than screening. The invention provides for a library of antibodies, or other ligand or receptor binding oligo- or polypeptides, to be displayed on the surface of phage. The invention requires that the phage are rendered non-infectious by modification of a minor coat protein required for infectivity. Phage which display oligo- or polypeptides with high affinity for a target ligand or receptor are selected from a library by conferring on them the ability to be propagated. The invention provides that phage displaying oligo- or polypeptides with lower target affinity are not propagated. Infectivity is conferred by a substance comprising the target ligand or receptor linked to a portion of the phage coat protein which is required for infectivity.
The main advantage over existing methods offered by the present invention is that it can be carried out in a continuous fashion. In this regard, it mimics the system of clonal selection used by the immune system in antibody optimization. The present invention provides a system which is well-suited to affinity maturation of antibodies in multiple rounds of mutation and selection. Even at a single step, the present invention provides an enormous simplification over existing methods since it provides for propagation of only binding variants, thus obviating the requirement for any chromatography or "panning" step. Extremely large libraries can, in principle, be screened. Furthermore, single binding events are detected, giving the method which is the subject of the present invention a very high sensitivity. Finally, the invention is not restricted to antibody selection; it applies equally to any oligo- or polypeptide which interacts with a target ligand or receptor.